Methods and compositions for monitoring and risk prediction in cardiorenal syndrome

ABSTRACT

The present invention relates to methods and compositions for monitoring, diagnosis, prognosis, and determination of treatment regimens in subjects. In particular, the invention relates to methods and compositions selected to monitor cardiorenal syndrome using assays that detect NGAL, preferably together with assays that detect natriuretic peptides such as BNP. Such methods and compositions can provide early indications of a deterioration in cardiorenal syndrome status, including prognosis regarding mortality and worsening renal function.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/541,232, filed on Jul. 3, 2012, which is a divisional applicationof U.S. application Ser. No. 13/152,009, filed on Jun. 2, 2011, now U.S.Pat. No. 8,283,128, which is a divisional of Ser. No. 12/909,654, filedon Oct. 21, 2010, now issued as U.S. Pat. No. 7,985,560 which is adivisional of Ser. No. 11/940,111, filed Nov. 14, 2007, now issued asU.S. Pat. No. 7,842,472, which in turn claims benefit under 35 U.S.C.§119(e) of U.S. Application No. 60/891,342 filed Feb. 23, 2007 and U.S.Application No. 60/859,137 filed Nov. 14, 2006, each of which isincorporated by reference herein for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which was originallyfiled on Jul. 22, 2011 in parent application Ser. No. 13/152,009, andwhich was submitted in ASCII format via EFS-Web and is herebyincorporated by reference in its entirety. Said ASCII copy, created onJul. 20, 2011, is named 36671-779-402seqlist.txt and is 3 Kilobytes insize.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for monitoringcardiorenal syndrome, and the heart failure and renal dysfunctionunderlying cardiorenal syndrome.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

The term “cardiorenal syndrome” refers to a physiologic relationshipbetween the heart and kidney that manifests as a tight coordinationbetween renal and cardiac functions in subjects suffering from heartfailure. While the syndrome is poorly understood, a feedback loopamongst neurohormonal systems (and, in particular, the natriureticpeptides), inflammatory responses, and structurally and functionallyimpaired organs has been implicated, creating a cycle of worseningcardiac and renal functions. A recent discussion of cardiorenal syndromemay be found in Francis, “Acute decompensated heart failure: Thecardiorenal syndrome,” Clev. Clinic J. Med. 73(S2): S8-S13, 2006.

In heart failure patients, the onset of renal dysfunction has proved astrong risk factor for mortality. In fact, an increased risk is signaledeven at serum creatinine levels >1.3 mg/dL and estimated creatinineclearance values ≦60 to 70 mL/min, values that can fall within “normal”values for each of these parameters. Although renal dysfunction predictsall-cause mortality, it is most predictive of death from progressiveheart failure, which suggests that it is a manifestation of and/orexacerbating factor for left ventricular dysfunction. And worseningrenal function may be even more important than baseline renal functionfor predicting adverse outcomes. In one multicenter study, a serumcreatinine increase of ≧0.3 mg/dL had a sensitivity of 65% andspecificity of 81% for predicting in-hospital mortality. Gottlieb etal., “The prognostic importance of different definitions of worseningrenal function in congestive heart failure,” J. Card. Fail. 8: 136-141,2002. Other studies have reported that deteriorating renal function isassociated with a longer length of stay, an increased risk of deathwithin 6 months after discharge, and a 33% increased risk for hospitalreadmission.

In addition, numerous studies have demonstrated that a variety of heartfailure therapies may actually worsen renal function, triggering adeterioration in the cardiorenal axis. For example, certain diureticshave been associated with worsening renal function, especially in thepresence of ACE inhibitors, and high diuretic doses have been associatedwith increased mortality rates. This is often thought to result from“diuretic resistance,” e.g., a failure to excrete at least 90 mmol ofsodium within 72 hours of a 160 mg oral furosemide dose given twicedaily, which necessitates increasing diuretic dosage. Whatever thecause, the resulting volume overload is poorly tolerated and a frequentcause of hospital admission in patients with heart failure.

In patients exhibiting worsening renal function, volume overload, anddiuretic refractoriness, the management of cardiorenal disease can beextremely difficult. Positive inotropic agents (including dobutamine,phosphodiesterase inhibitors, and levosimendan) may facilitate adiuresis with preservation or improvement in renal function. Althoughdopamine also is used because of its presumed ability to improve renalblood flow, this effect is severely limited in advanced heart failure.Intravenous vasodilators can improve hemodynamics, but often will notimprove renal function.

In recent years, natriuretic peptide measurement has dramaticallychanged the diagnosis and management of cardiac diseases, includingheart failure and the acute coronary syndromes. In particular, B-typenatriuretic peptide (BNP, human precursor Swiss-Prot P16860), andvarious related polypeptides arising from the common precursor proBNP,have been used to diagnose heart failure, determine its severity, andestimate prognosis. In addition, BNP and its related polypeptides havebeen demonstrated to provide diagnostic and prognostic information inunstable angina, non-ST-elevation myocardial infarction, andST-elevation myocardial infarction.

In contrast, current diagnostic tests for renal dysfunction, such asserum creatinine or cystatin C measurements, can be misleading to theclinician. While it is preferred that aggressive treatment begin at theearliest indication of renal dysfunction, these tests may only becomeabnormal days after the original insult. A large proportion of the renalmass may be damaged before any biochemical evidence of renal dysfunctionis appreciated, as the rise of serum creatinine may not be evidentbefore 50% of the glomerular filtration rate is lost. No surprisinglyperhaps, it has been reported that about two thirds of the patientsadmitted for acute heart failure have inadequate glomerular filtrationrates or creatinine clearance, despite relatively normal serumcreatinine levels. Recently, NGAL (also known as neutrophilgelatinase-associated lipocalin, human precursor Swiss-Prot P80188) hasbeen proposed as a new early marker for acute renal injury, with reportsof increased levels of NGAL from acute renal injury detectable in bothurine and blood within two hours of the insult. See, e.g., WO04088276;WO05121788; WO06066587. The use of NGAL as a risk marker in the contextof heart failure, renal dysfunction, or cardiorenal syndrome has notbeen described.

BRIEF SUMMARY OF THE INVENTION

It is an object of the invention to provide methods and compositions fordiagnosis, prognosis, and determination of treatment regimens insubjects suffering from renal dysfunction, heart failure, andcardiorenal syndrome. In various aspects, the present invention providesmethods for assessing risk of worsening renal function in the context ofheart failure; methods for assigning risk of mortality in the context ofrenal dysfunction, methods of monitoring cardiorenal syndrome; andvarious devices and kits adapted to perform such methods.

In a first aspect, the present invention relates to methods forassigning a risk of worsening renal function to a patient suffering fromheart failure. These methods comprise performing an assay method thatconfigured to detect NGAL (and/or one or more markers related thereto,as that term is defined herein) on a body fluid sample obtained from asubject suffering from heart failure. The assay result (referred toherein as an “NGAL assay result,” which may be expressed in the form ofan NGAL concentration) is compared to a baseline NGAL result, and anincreased risk of worsening renal function is assigned to the subjectwhen the NGAL assay result is above the baseline, relative to a riskassigned when the NGAL assay result is below the baseline. In thealternative, a decreased risk of worsening renal function is assigned tothe subject when the NGAL assay result is below the baseline, relativeto a risk assigned when the NGAL assay result is above than thebaseline.

In a related aspect, the present invention relates to methods forassigning a risk of an adverse outcome to a patient suffering from heartfailure. These methods comprise performing an assay method configured todetect NGAL (and/or one or more markers related thereto, as that term isdefined herein) on a body fluid sample obtained from a subject sufferingfrom heart failure. The assay result (referred to herein as an “NGALassay result,” which may be expressed in the form of an NGALconcentration) is compared to a baseline NGAL result, and an increasedrisk of one or more adverse outcomes is assigned to the subject when theNGAL assay result is above the baseline, relative to a risk assignedwhen the NGAL assay result is below the baseline. In the alternative, adecreased risk of one or more adverse outcomes is assigned to thesubject when the NGAL assay result is below the baseline, relative to arisk assigned when the NGAL assay result is above the baseline. Asdescribed hereinafter, preferred adverse outcomes are mortality and/oran acute cardiac event requiring immediate medical care. Preferred acutecardiac events are symptoms resulting from heart failure or an acutecoronary syndrome, such a chest pain and/or dyspnea.

A variety of methods may be used by the skilled artisan to arrive at adesired baseline. In certain preferred embodiments, the baseline NGALresult is determined from an earlier NGAL assay result obtained from thesame subject. That is, the change in NGAL concentration may be observedover time, and an increased NGAL concentration provides an indication ofworsening renal function in the heart failure patient.

In alternative embodiments, the baseline NGAL result is determined froma population of subjects suffering from heart failure as describedhereinafter, and is based on an NGAL result that can provide anacceptable level of specificity and sensitivity in separating thepopulation into a “first” subpopulation (e.g., having an increased riskof worsening (or improving) renal function or an increased risk of anadverse outcome) relative to the remaining “second” subpopulation. Asdiscussed herein, a preferred baseline NGAL result separates this firstand second population by one or more of the following measures of testaccuracy:

an odds ratio of at least about 2 or more or about 0.5 or less, morepreferably at least about 3 or more or about 0.33 or less, still morepreferably at least about 4 or more or about 0.25 or less, even morepreferably at least about 5 or more or about 0.2 or less, and mostpreferably at least about 10 or more or about 0.1 or less.at least 75% sensitivity, combined with at least 75% specificity;a ROC curve area of at least 0.6, more preferably 0.7, still morepreferably at least 0.8, even more preferably at least 0.9, and mostpreferably at least 0.95; and/ora positive likelihood ratio (calculated as sensitivity/(1-specificity))of at least 5, more preferably at least 10, and most preferably at least20; ora negative likelihood ratio (calculated as (1-sensitivity)/specificity)of less than or equal to 0.3, more preferably less than or equal to 0.2,and most preferably less than or equal to 0.1. The term “about” in thiscontext refers to +/−5% of a given measurement.

The present methods preferably assign a “near-term” risk of worseningrenal function and/or risk of an adverse outcome. By “near term” ismeant within 30 days. As described hereinafter, the methods preferablyassign a risk of worsening renal function and/or mortality within 7days, more preferably within 5 days, and still more preferably within 3days.

Preferred assay methods comprise performing an immunoassay that detectsNGAL. Antibodies for use in such assays will specifically bind NGAL, andmay optionally also bind one or more polypeptides that are “related”thereto, as described hereinafter with regard to related markers. Suchimmunoassays may comprise contacting said body fluid sample with a solidphase comprising antibody that detects NGAL, and detecting binding tosaid antibody, although assay formats that do not require the use of asolid phase are known in the art. Preferably, the body fluid sample isselected from the group consisting of urine, blood, serum, and plasma.

In addition to NGAL assay results, additional variables may be includedin the methods for assigning a risk of worsening renal function and/orrisk of an adverse outcome described herein. As described in additionaldetail hereinafter, assays that detect various markers (bothsubject-derived and physical characteristics) may be combined, includingassays that detect various natriuretic peptides such as BNP, NT-proBNP,and proBNP; markers related to inflammation such as myeloperoxidase,soluble FLT-1, C-reactive protein, and placental growth factor; markersrelated to cardiac damage such as cardiac troponins and CK-MB; markersof renal damage such as serum creatinine, creatinine clearance rates,cystatin C, and glomerular filtration rates; and variables such as urineoutput levels, age, the presence or absence of various cardiovascularrisk factors such as diabetes, hypertension, body mass, smoking status;etc. In these “multiple marker” methods, the patient's risk of worseningrenal function or risk of an adverse outcome is assigned based both oncomparing the NGAL assay result to the baseline NGAL result as describedabove, and on one or more of these additional variables. In many cases,these additional variables can be used in a manner analogous to NGAL, inthat baseline results can be established for these markers as describedherein for comparison of a test result measured on a body fluid sample.In other cases, such as sex, cardiovascular risk factors, etc., thevariables can be dichotomized for analysis of risk. For example,different NGAL baselines may be established for different age groups,and/or based on sex of the subject.

In methods where multiple assays are performed on body fluids, thevarious assays can be performed on the same or different body fluidsamples. For example, NGAL may be measured in a urine sample and BNP maybe measured in a plasma sample; or NGAL may be measured in a plasmasample and cystatin C measured in a different plasma sample.

In another aspect, the present invention relates to methods forassigning a risk of mortality to a patient suffering from renaldysfunction. These methods comprise performing an assay method thatconfigured to detect NGAL (and/or one or more markers related thereto,as that term is defined herein) on a body fluid sample obtained from asubject suffering from renal dysfunction. The assay result (referred toherein as an “NGAL assay result,” which may be expressed in the form ofan NGAL concentration) is compared to a baseline NGAL result, and anincreased mortality risk is assigned to the subject when the NGAL assayresult is above the baseline, relative to a risk assigned when the NGALassay result is below the baseline. In the alternative, a decreasedmortality risk is assigned to the subject when the NGAL assay result isbelow the baseline, relative to a risk assigned when the NGAL assayresult is above than the baseline.

As discussed above, a variety of methods may be used by the skilledartisan to arrive at a desired baseline. In certain preferredembodiments, the baseline NGAL result is determined from an earlier NGALassay result obtained from the same subject. That is, the change in NGALconcentration may be observed over time, and an increased NGALconcentration provides an indication of worsening renal function in theheart failure patient.

In alternative embodiments, the baseline NGAL result is determined froma population of subjects suffering from renal dysfunction as describedhereinafter, and is based on an NGAL result that can provide anacceptable level of specificity and sensitivity in separating thepopulation into a “first” subpopulation having an increased (ordecreased) mortality risk relative to the remaining “second”subpopulation. As discussed herein, a preferred baseline NGAL resultseparates this first and second population by one or more of thefollowing measures of test accuracy:

an odds ratio of at least about 2 or more or about 0.5 or less, morepreferably at least about 3 or more or about 0.33 or less, still morepreferably at least about 4 or more or about 0.25 or less, even morepreferably at least about 5 or more or about 0.2 or less, and mostpreferably at least about 10 or more or about 0.1 or less.at least 75% sensitivity, combined with at least 75% specificity;a ROC curve area of at least 0.6, more preferably 0.7, still morepreferably at least 0.8, even more preferably at least 0.9, and mostpreferably at least 0.95; and/ora positive likelihood ratio (calculated as sensitivity/(1-specificity))of at least 5, more preferably at least 10, and most preferably at least20; ora negative likelihood ratio (calculated as (1-sensitivity)/specificity)of less than or equal to 0.3, more preferably less than or equal to 0.2,and most preferably less than or equal to 0.1. The term “about” in thiscontext refers to +/−5% of a given measurement.

The present methods preferably assign a “near-term” mortality risk. By“near term” is meant within 30 days. As described hereinafter, themethods preferably assign a mortality risk within 7 days, morepreferably within 5 days, and still more preferably within 3 days.

As is also described above, preferred assay methods comprise performingan immunoassay that detects NGAL. Antibodies for use in such assays canspecifically bind NGAL, and may optionally also bind one or morepolypeptides that are “related” thereto. Such immunoassays may comprisecontacting said body fluid sample with a solid phase comprising antibodythat detects NGAL, and detecting binding to said antibody, althoughassay formats that do not require the use of a solid phase are known inthe art. Preferably, the body fluid sample is selected from the groupconsisting of urine, blood, serum, and plasma.

In addition to NGAL assay results, additional variables may be includedin the methods for assigning a mortality risk in the context of renaldysfunction. As described in additional detail hereinafter, assays thatdetect various markers (both subject-derived and physicalcharacteristics) may be combined, including assays that detect variousnatriuretic peptides such as BNP, NT-proBNP, and proBNP; markers relatedto inflammation such as myeloperoxidase, soluble FLT-1, C-reactiveprotein, and placental growth factor; markers related to cardiac damagesuch as cardiac troponins and CK-MB; markers of renal damage such asserum creatinine, creatinine clearance rates, cystatin C, and glomerularfiltration rates; and variables such as urine output levels, age, thepresence or absence of various cardiovascular risk factors such asdiabetes, hypertension, body mass, smoking status; etc. In these“multiple marker” methods, the patient's mortality risk is assignedbased both on comparing the NGAL assay result to the baseline NGALresult as described above, and on one or more of these additionalvariables. In many cases, these additional variables can be used in amanner analogous to NGAL, in that baseline results can be establishedfor these markers as described herein for comparison of a test resultmeasured on a body fluid sample. In other cases, such as sex,cardiovascular risk factors, etc., the variables can be dichotomized foranalysis of risk. For example, different NGAL baselines may beestablished for different age groups, and/or based on sex of thesubject.

As noted above, the various assays can be performed on the same ordifferent body fluid samples. For example, NGAL may be measured in aurine sample and BNP may be measured in a plasma sample; or NGAL may bemeasured in a plasma sample and cystatin C measured in a differentplasma sample.

In still another aspect, the present invention relates to methods formonitoring cardiorenal syndrome in a patient. These methods compriseperforming an assay method that configured to detect NGAL and an assaymethod configured to detect one or more of BNP, NT-proBNP and proBNP(and/or one or more markers related thereto in each case, as that termis defined herein) on a body fluid sample obtained from a subject. Eachof the assay results (referred to herein as an “NGAL assay result,”which may be expressed in the form of an NGAL concentration, and a“natriuretic peptide result,” which may be expressed in the form of aconcentration of one or more of BNP, NT-proBNP and proBNP) is comparedto a corresponding baseline result—that is, a baseline NGAL result and abaseline natriuretic peptide result. A worsening cardiorenal syndromestatus may be assigned to the patient if either or both of the assayresults are greater than the corresponding baseline result. In thealternative, an improving cardiorenal syndrome status may be assigned tothe patient if either or both of the assay results are less than thecorresponding baseline result.

As in the previous aspects, a variety of methods may be used by theskilled artisan to arrive at each desired baseline values. In certainpreferred embodiments, the baseline result is determined from an earlierassay result obtained from the same subject. In alternative embodiments,the baseline result is determined from a population of subjects, and isbased on an assay result that can provide an acceptable level ofspecificity and sensitivity in separating the population into a “first”subpopulation having a worsening (or improving) cardiorenal syndromestatus relative to the remaining “second” subpopulation.

As discussed herein, a preferred baseline result separates this firstand second population by one or more of the following measures of testaccuracy:

an odds ratio of at least about 2 or more or about 0.5 or less, morepreferably at least about 3 or more or about 0.33 or less, still morepreferably at least about 4 or more or about 0.25 or less, even morepreferably at least about 5 or more or about 0.2 or less, and mostpreferably at least about 10 or more or about 0.1 or less.at least 75% sensitivity, combined with at least 75% specificity;a ROC curve area of at least 0.6, more preferably 0.7, still morepreferably at least 0.8, even more preferably at least 0.9, and mostpreferably at least 0.95; and/ora positive likelihood ratio (calculated as sensitivity/(1-specificity))of at least 5, more preferably at least 10, and most preferably at least20; ora negative likelihood ratio (calculated as (1-sensitivity)/specificity)of less than or equal to 0.3, more preferably less than or equal to 0.2,and most preferably less than or equal to 0.1. The term “about” in thiscontext refers to +/−5% of a given measurement.

The present methods preferably assign a “near-term” change incardiorenal syndrome status. By “near term” is meant within 30 days. Asdescribed hereinafter, the methods preferably assign a mortality riskwithin 7 days, more preferably within 5 days, and still more preferablywithin 3 days.

As is also described above, preferred assay methods comprise performingan immunoassay that detects NGAL and/or an immunoassay that detects oneor more of BNP, NT-proBNP and proBNP. Antibodies for use in such assayscan specifically bind to the intended target(s) of the assay, and mayoptionally also bind one or more polypeptides that are “related”thereto. Such immunoassays may comprise contacting said body fluidsample with a solid phase comprising antibody that detects the intendedtarget(s), and detecting binding to said antibody, although assayformats that do not require the use of a solid phase are known in theart. Preferably, the body fluid sample is selected from the groupconsisting of urine, blood, serum, and plasma.

Additional variables may be included in the methods for assigning amortality risk in the context of renal dysfunction. As described inadditional detail hereinafter, assays that detect various markers (bothsubject-derived and physical characteristics) may be combined, includingassays that detect various natriuretic peptides other than BNP,NT-proBNP, and proBNP; markers related to inflammation such asmyeloperoxidase, soluble FLT-1, C-reactive protein, and placental growthfactor; markers related to cardiac damage such as cardiac troponins andCK-MB; markers of renal damage such as serum creatinine, creatinineclearance rates, cystatin C, and glomerular filtration rates; andvariables such as urine output levels, age, the presence or absence ofvarious cardiovascular risk factors such as diabetes, hypertension, bodymass, smoking status; etc.

As noted above, the various assays can be performed on the same ordifferent body fluid samples. For example, NGAL may be measured in aurine sample and BNP may be measured in a plasma sample; or NGAL may bemeasured in a plasma sample and BNP measured in a different plasmasample.

In various related aspects, the present invention relates to devices andkits for performing the methods described herein. Suitable kits comprisereagents sufficient for performing at least one of the described assays,together with instructions for performing the described baselinecomparisons. In the case of assigning a risk of worsening renalfunction, such instructions can include instructions for obtaining thedescribed NGAL baseline result, and/or for assigning a risk of worseningrenal function based on the results of the comparison, and such kits canoptionally include comparable reagents and instructions for measuringand using additional variables such as BNP, NT-proBNP, and proBNP, etc.,as described above. In the case of assigning a risk of mortality, suchinstructions can include instructions for obtaining the described NGALbaseline result, and/or for assigning a risk of mortality based on theresults of the comparison, and such kits can optionally includecomparable reagents and instructions for measuring and using additionalvariables such as BNP, NT-proBNP, and proBNP, etc., as described above.In the case of monitoring cardiorenal syndrome, such instructions caninclude instructions for obtaining the described NGAL and natriureticpeptide baseline results, and/or for assigning a change in cardiorenalsyndrome status based on the results of the comparisons, and such kitscan optionally include comparable reagents and instructions formeasuring and using additional variables such as various natriureticpeptides other than BNP, NT-proBNP, and proBNP, etc., as describedabove.

In certain embodiments, reagents for performing such assays are providedin an assay device, and such assay devices may be included in such akit. Preferred reagents comprise one or more solid phase antibodies, thesolid phase antibody comprising antibody that detects the intendedtarget(s) bound to a solid support. In the case of sandwichimmunoassays, such reagents can also include one or more detectablylabeled antibodies, the detectably labeled antibody comprising antibodythat detects the intended target(s) bound to a detectable label.Additional optional elements that may be provided as part of an assaydevice are described hereinafter. A preferred assay device comprisesreagents for performing an assay that detects NGAL (and/or one or morerelated markers), and reagents for performing an assay that detects oneor more of BNP, NT-proBNP, and proBNP (and/or one or more relatedmarkers).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for diagnosis,prognosis, and determination of treatment regimens in subjects sufferingfrom renal dysfunction, heart failure, and cardiorenal syndrome.

Congestive heart failure (CHF) is a fatal disease with a 5-yearmortality rate that rivals the most deadly malignancies. For example, inthe Framingham Heart Study, median survival after the onset of heartfailure was 1.7 years in men and 3.2 years in women. Overall, 1-year and5-year survival rates were 57% and 25% in men and 64% and 38% in women,respectively. Moreover, a person age 40 or older has a one-in-fivelifetime chance of developing congestive heart failure. Moreover, thebody relies on a coupling of renal function to cardiac function, acharacteristic sometimes referred to as the “cardiorenal axis.” Whilethe precise pathophysiological mechanisms underlying this relationshipbetween the heart and kidneys are still emerging, it has now becomeclear that the co-existence renal dysfunction and heart failure signalsan extremely bad prognosis.

The appropriate treatments given to patients suffering from renaldysfunction and heart failure can be large and diverse. Unfortunately,many treatments given for various diseases may adversely affect thecardiorenal axis. For example, diuretics are often given to reduce theincreased fluid load characteristic of heart failure, and non-steroidalanti-inflammatory drugs (NSAIDs) are often prescribed to elderlysubjects. Each of these drugs increase the risk of damage to both theheart and kidneys.

The present invention provides methods and compositions that may be usedto monitor the cardiorenal axis in an improved manner. By monitoring theends of this axis using markers that provide an early indication thatdamage may follow, treatments may be balanced to provide benefit to thepatient while at the same time preserving cardiorenal status. Forexample, treatments known to damage the heart and/or kidney may betemporarily removed in the face of a worsening cardiorenal syndromestatus or worsening renal function. Alternatively, or in addition,palliative treatments may be selected and initiated in an attempt toimprove the function of the heart or kidney, while observing the overalleffect on the cardiorenal syndrome status of the patient.

The term “renal dysfunction” as used herein refers to a reduced level ofkidney function, relative to normal, and is indicated by a serum orplasma creatinine level 1.4 mg/dL or greater, a creatinine clearancerate of 60 mL/min/1.73 m² or less, a serum or plasma cystatin C level of1 mg/L or greater, or the presence of chronic kidney disease as definedby the National Kidney Foundation in “National Kidney Foundation. KidneyDisease Outcome Quality Initiative (K/DOQI) clinical practice guidelinesfor chronic kidney disease: evaluation, classification, andstratification,” Am. J. Kidney Dis. 39(Suppl 1):S1-S266, 2002, which ishereby incorporated by reference in its entirety. Chronic kidney diseaseincludes persistent kidney damage, as reflected by a glomerularfiltration rate (GFR) of less than 60.0 mL/minute/1.73 m² ofbody-surface area for more than three months. Included are patients withend-stage renal disease, as defined by a GFR of less than 10.0mL/minute/1.73 m². Because practical limitations exist in measuring GFRdirectly, especially in acutely ill patients, clinical variables may beused to estimate the GFR. See, e.g., Levey et al., “A more accuratemethod to estimate glomerular filtration rate from serum creatinine: anew prediction equation,” Ann. Intern. Med. 130: 461-70, 1999, which ishereby incorporated by reference in its entirety.

Preferred subjects having renal dysfunction are those having severerenal dysfunction (defined herein as serum or plasma creatinine >2.5mg/dL, serum or plasma cystatin C>2 mg/L, or creatinine clearance ratesof <25 mL/minute/1.73 m²), more preferred subjects have moderate renaldysfunction (defined herein as serum or serum or plasma creatinine of1.9 to 2.5 mg/dL, serum or plasma cystatin C of 2 to 1.5 mg/L, orcreatinine clearance rates of 25-30 mL/minute/1.73 m²), and mostpreferred subjects have mild renal dysfunction (defined herein as serumor plasma creatinine of 1.4 to 2.25 mg/dL, serum or plasma cystatin C of1 to 1.5 mg/L, or creatinine clearance rates of 30-60 mL/minute/1.73m²).

The term “risk of worsening renal function” as used herein refers toassignment of a particular prognosis—a likelihood that a subject willsuffer, most preferably in the short term, from deterioration of one ormore measures of kidney function selected from the group consisting ofserum or plasma creatinine levels, creatinine clearance rates, serum orplasma cystatin C levels, chronic kidney disease stage (as defined bythe National Kidney Foundation in “National Kidney Foundation. KidneyDisease Outcome Quality Initiative (K/DOQI) clinical practice guidelinesfor chronic kidney disease: evaluation, classification, andstratification,” Am. J. Kidney Dis. 39(Suppl 1): S1-S266, 2002), stageof renal dysfunction, and glomerular filtration rate, or, in theextreme, will die (that is, a likelihood of mortality in the subject).

As described herein, the present invention relates in part to assigninga risk of worsening renal function based, at least in part, on theresult of an assay that detects NGAL performed on a body fluid sampleobtained from a subject. If the sample tested is obtained from thesubject at a time t, the phrase “the short term” refers to a 7-day (168hour) period measured from time t. Thus, the risk is a likelihood thatthe subject will suffer from deterioration of one or more of thesemeasures of kidney function, or will die, in a window beginning at timet and ending 168 hours later. More preferably, the risk is a likelihoodthat the subject will suffer from deterioration of one or more of thesemeasures of kidney function, or will die, in a 96 hour window beginningat time t, and most preferably the risk is a likelihood that the subjectwill suffer from deterioration of one or more of these measures ofkidney function, or a likelihood that the subject will die, in a windowof between 48 and 84 hours beginning at time t.

The term “deterioration” as used herein refers to a change in aparameter at a later time, relative to a measure of the same parameterearlier in the same subject, and is the opposite of “improvement.” Thus,the terms “deterioration of serum or plasma creatinine levels” and“deterioration of serum or plasma cystatin C levels” as used hereinrefer to an increase in the creatinine or cystatin C assay result in asample obtained from a subject at a time later than time t, relative tothat measured in the subject at time t. Similarly, “deterioration ofcreatinine clearance rates” as used herein refers to a later decrease increatinine clearance rate for the subject, relative to that measured attime t. “Deterioration in chronic kidney disease stage” as used hereinrefers to a later change to a higher stage in the subject, relative tothat observed at time t. “Deterioration of stage of renal dysfunction”as used herein refers to a later change in the subject from mild tomoderate or severe, or a change from moderate to severe, relative to thestage of renal dysfunction observed at time t. Finally, “deteriorationof glomerular filtration rate” as used herein refers to a later decreasein GFR in the subject, relative to that observed at time t.

The term “monitoring cardiorenal syndrome” refers to observing a patientdiagnosed with heart failure for changes in coordination between renaland cardiac functions as measured by a change in renal function, cardiacoutput function, or both. A “change in cardiorenal syndrome status” asused herein refers to assignment of a particular prognosis—thelikelihood that a subject diagnosed with heart failure will suffer, mostpreferably in the short term, from a change (improvement ordeterioration) of one or more measures of renal or cardiac outputfunction, or, in the extreme, the likelihood that a subject diagnosedwith heart failure will die (that is, a likelihood of mortality in thesubject). As described herein, NGAL assays may be used to predictchanges in renal function, the future onset of renal dysfunction, and/orworsening of renal function. Similarly, as described herein, assays forBNP and its related polypeptides may be used to predict changes incardiac output function in heart failure. By combining thesemeasurements, the skilled artisan can initiate or alter treatment of asubject in advance of the subject exhibiting outward clinical signs ofdeteriorating cardiorenal syndrome status.

The term “cardiac output function” as used herein refers to one or morecharacteristics of heart function that are affected in subjectsdiagnosed with heart failure. Such characteristics include one or moreof: dyspnea (at rest or exertional), orthopnea, pulmonary edema, SaO₂level, dizziness or syncope, chest pain, systolic blood pressure,hypoperfusion, edema, NYHA grade, compensation status (that is, a changefrom compensated to decompensated, or vice versa), ejection fraction,end-diastolic function, end-systolic function, ventricular filling, andflow across the mitral valve. These characteristics, and methods fortheir assessment, are well known in the art. See, e.g., Harrison'sPrinciples of Internal Medicine, 16^(th) ed., McGraw-Hill, 2005, pages1361-1377, which is hereby incorporated by reference in its entirety. Asdiscussed above, “deterioration” as used herein refers to a change in aparameter at a later time, relative to a measure of the same parameterearlier in the same subject. Thus, “deterioration in cardiac outputfunction” refers to a change in one or more of these characteristicsindicative of a clinical worsening of heart failure in a subject.

The term “marker” as used herein refers to proteins, polypeptides,glycoproteins, proteoglycans, lipids, lipoproteins, glycolipids,phospholipids, nucleic acids, carbohydrates, etc. or small molecules tobe used as targets for screening test samples obtained from subjects.“Proteins or polypeptides” used as markers in the present invention arecontemplated to include any fragments thereof, in particular,immunologically detectable fragments. Markers can also include clinical“scores” such as a pre-test probability assignment, a pulmonaryhypertension “Daniel” score, an NIH stroke score, a Sepsis Score ofElebute and Stoner, a Duke Criteria for Infective Endocarditis, aMannheim Peritonitis Index, an “Apache” score, etc.

The term “related marker” as used herein refers to one or more fragmentsof a particular marker or its biosynthetic parent that may be detectedas a surrogate for the marker itself or as independent markers. Forexample, human BNP is derived by proteolysis of a 108 amino acidprecursor molecule, referred to hereinafter as BNP₁₋₁₀₈. Mature BNP, or“the BNP natriuretic peptide,” or “BNP-32” is a 32 amino acid moleculerepresenting amino acids 77-108 of this precursor, which may be referredto as BNP₇₇₋₁₀₈. The remaining residues 1-76 are referred to hereinafteras BNP₁₋₇₆, and are also known as “NT-proBNP.” Additionally, relatedmarkers may be the result of covalent modification of the parent marker,for example by oxidation of methionine residues, ubiquitination,cysteinylation, nitrosylation (e.g., containing nitrotyrosine residues),halogenation (e.g., containing chlorotyrosine and/or bromotyrosineresidues), glycosylation, complex formation, differential splicing, etc.Preferred related markers are “immunologically detectable,” meaning afragment of a particular marker or its biosynthetic parent thatcomprises at least one epitope (antigenic site on the polypeptide towhich an antibody binds) that is also present in the marker or itsbiosynthetic parent. Preferred immunologically detectable fragmentscomprise at least 8 contiguous residues of the marker or itsbiosynthetic parent.

The sequence of the 108 amino acid BNP precursor pro-BNP (BNP₁₋₁₀₈) isas follows, with mature BNP (BNP₇₇₋₁₀₈) underlined:

(SEQ ID NO: 1) HPLGSPGSAS DLETSGLQEQ RNHLQGKLSE  50LQVEQTSLEP LQESPRPTGV WKSREVATEG IRGHRKMVLY TLRAPRSPKM 100VQGSGCFGRK MDRISSSSGL GCKVLRRH. 108

BNP₁₋₁₀₈ is synthesized as a larger precursor pre-pro-BNP having thefollowing sequence (with the “pre” sequence shown in bold):

(SEQ ID NO: 2) MDPQTAPSRA LLLLLFLHLA FLGGRSHPLG  50SPGSAS DLET SGLQEQRNHL QGKLSELQVE QTSLEPLQES PRPTGVWKSR 100EVATEGIRGH RKMVLYTLRA PRSPKMVQGS GCFGRKMDRI SSSSGLGCKV 134 LRRH.

While mature BNP itself may be used as a marker in the presentinvention, the prepro-BNP, BNP₁₋₁₀₈ and BNP₁₋₇₆ molecules representBNP-related markers that may be measured either as surrogates for matureBNP or as markers in and of themselves. In addition, one or morefragments of these molecules, including BNP-related polypeptidesselected from the group consisting of BNP₇₇₋₁₀₆, BNP₇₉₋₁₀₆, BNP₇₆₋₁₀₇,BNP₆₉₋₁₀₈, BNP₇₉₋₁₀₈, BNP₈₀₋₁₀₈, BNP₈₁₋₁₀₈, BNP₈₃₋₁₀₈, BNP₃₉₋₈₆,BNP₅₃₋₈₅, BNP₆₆₋₉₈, BNP₃₀₋₁₀₃, BNP₁₁₋₁₀₇, BNP₉₋₁₀₆, and BNP₃₋₁₀₈, mayalso be present in circulation. In addition, natriuretic peptidefragments, including BNP fragments, may comprise one or more oxidizablemethionines, the oxidation of which to methionine sulfoxide ormethionine sulfone produces additional BNP-related markers. See, e.g.,U.S. patent Ser. No. 10/419,059, filed Apr. 17, 2003, which is herebyincorporated by reference in its entirety including all tables, figuresand claims. Moreover, assays may intentionally use proteolysis togenerate fragments of a marker, which may then be measured. See, e.g.,Goetze et al., “Quantification of pro-B-type natriuretic peptide and itsproducts in human plasma by use of an analysis independent of precursorprocessing,” Clin. Chem. 48: 1035-42, 2002. “Related markers” to each ofthe markers described herein may be identified and used in an analogousfashion to that described above for BNP.

Because production of marker fragments is an ongoing process that may bea function of, inter alia, the elapsed time between onset of an eventtriggering marker release into the tissues and the time the sample isobtained or analyzed; the elapsed time between sample acquisition andthe time the sample is analyzed; the type of tissue sample at issue; thestorage conditions; the quantity of proteolytic enzymes present; etc.,it may be necessary to consider this degradation when both designing anassay for one or more markers, and when performing such an assay, inorder to provide an accurate prognostic or diagnostic result. Inaddition, individual antibodies that distinguish amongst a plurality ofmarker fragments may be individually employed to separately detect thepresence or amount of different fragments. The results of thisindividual detection may provide a more accurate prognostic ordiagnostic result than detecting the plurality of fragments in a singleassay. For example, different weighting factors may be applied to thevarious fragment measurements to provide a more accurate estimate of theamount of natriuretic peptide originally present in the sample.

The failure to consider the degradation fragments that may be present ina clinical sample may have serious consequences for the accuracy of anydiagnostic or prognostic method. Consider for example a simple case,where a sandwich immunoassay is provided for BNP, and a significantamount (e.g., 50%) of the biologically active BNP that had been presenthas now been degraded into an inactive form. An immunoassay formulatedwith antibodies that bind a region common to the biologically active BNPand the inactive fragment(s) will overestimate the amount ofbiologically active BNP present in the sample by 2-fold, potentiallyresulting in a “false positive” result. Overestimation of thebiologically active form(s) present in a sample may also have seriousconsequences for patient management. Considering the BNP example again,the BNP concentration may be used to determine if therapy is effective(e.g., by monitoring BNP to see if an elevated level is returning tonormal upon treatment). The same “false positive” BNP result discussedabove may lead the physician to continue, increase, or modify treatmentbecause of the false impression that current therapy is ineffective.

Likewise, it may be necessary to consider the complex state of one ormore markers described herein. For example, troponin exists in musclemainly as a “ternary complex” comprising three troponin polypeptides (T,I and C). But troponin I and troponin T circulate in the blood in formsother than the I/T/C ternary complex. Rather, each of (i) freecardiac-specific troponin I, (ii) binary complexes (e.g., troponin I/Ccomplex), and (iii) ternary complexes all circulate in the blood.Furthermore, the “complex state” of troponin I and T may change overtime in a patient, e.g., due to binding of free troponin polypeptides toother circulating troponin polypeptides. Immunoassays that fail toconsider the “complex state” of troponin may not detect all of thecardiac-specific isoform of interest.

Preferred assays are “configured to detect” a particular marker. That anassay is “configured to detect” a marker means that an assay cangenerate a detectable signal indicative of the presence or amount of aphysiologically relevant concentration of a particular marker ofinterest. Such an assay may, but need not, specifically detect aparticular marker (i.e., detect a marker but not some or all relatedmarkers). Because an antibody epitope is on the order of 8 amino acids,an immunoassay will detect other polypeptides (e.g., related markers) solong as the other polypeptides contain the epitope(s) necessary to bindto the antibody used in the assay. Such other polypeptides are referredto as being “immunologically detectable” in the assay, and would includevarious isoforms (e.g., splice variants). In the case of a sandwichimmunoassay, related markers must contain at least the two epitopesbound by the antibody used in the assay in order to be detected. TakingBNP₇₉₋₁₀₈ as an example, an assay configured to detect this marker mayalso detect BNP₇₇₋₁₀₈ or BNP₁₋₁₀₈, as such molecules may also containthe epitope(s) present on BNP₇₉₋₁₀₈ to which the assay antibody binds.However, such assays may also be configured to be “sensitive” to loss ofa particular epitope, e.g., at the amino and/or carboxyl terminus of aparticular polypeptide of interest as described in US2005/0148024, whichis hereby incorporated by reference in its entirety. As describedtherein, an antibody may be selected that would bind to the aminoterminus of BNP₇₉₋₁₀₈ such that it does not bind to BNP₇₇₋₁₀₈. Similarassays that bind BNP₃₋₁₀₈ and that are “sensitive” to loss of aparticular epitiope, e.g., at the amino and/or carboxyl terminus arealso described therein.

The methods described hereinafter utilize assays that detect BNP, NGAL,and/or their related markers. Such markers are referred to herein asbeing “subject-derived.” The term “subject-derived marker” as usedherein refers to protein, polypeptide, phospholipid, nucleic acid,prion, glycoprotein, proteoglycan, glycolipid, lipid, lipoprotein,carbohydrate, or small molecule markers that are expressed or producedby one or more cells of the subject. Additional markers may be used thatare derived not from the subject, but rather that are expressed bypathogenic or infectious organisms that are correlated with a particulardisease. Such markers are preferably protein, polypeptide, phospholipid,nucleic acid, prion, or small molecule markers that identify theinfectious diseases described above. These subject-derived markers aremeasured in a test sample, most preferably a body fluid sample.

The term “test sample” as used herein refers to a sample of bodily fluidobtained for the purpose of diagnosis, prognosis, or evaluation of asubject of interest, such as a patient. In certain embodiments, such asample may be obtained for the purpose of determining the outcome of anongoing condition or the effect of a treatment regimen on a condition.Preferred test samples include blood, serum, plasma, cerebrospinalfluid, urine, saliva, sputum, and pleural effusions. In addition, one ofskill in the art would realize that some test samples would be morereadily analyzed following a fractionation or purification procedure,for example, separation of whole blood into serum or plasma components.

As used herein, a “plurality” as used herein refers to at least two.Preferably, a plurality refers to at least 3, more preferably at least5, even more preferably at least 10, even more preferably at least 15,and most preferably at least 20. In particularly preferred embodiments,a plurality is a large number, i.e., at least 100.

The term “subject” as used herein refers to a human or non-humanorganism. Thus, the methods and compositions described herein areapplicable to both human and veterinary disease. Further, while asubject is preferably a living organism, the invention described hereinmay be used in post-mortem analysis as well. Preferred subjects are“patients,” i.e., living humans that are receiving medical care for adisease or condition. This includes persons with no defined illness whoare being investigated for signs of pathology.

The term “diagnosis” as used herein refers to methods by which theskilled artisan can estimate and/or determine whether or not a patientis suffering from a given disease or condition. The skilled artisanoften makes a diagnosis on the basis of one or more diagnosticindicators, i.e., a marker, the presence, absence, amount, or change inamount of which is indicative of the presence, severity, or absence ofthe condition. The term “diagnosis” does not refer to the ability todetermine the presence or absence of a particular disease with 100%accuracy, or even that a given course or outcome is more likely to occurthan not. Instead, the skilled artisan will understand that the term“diagnosis” refers to an increased probability that a certain disease ispresent in the subject.

Similarly, a prognosis is often determined by examining one or more“prognostic indicators.” These are markers, the presence or amount ofwhich in a patient (or a sample obtained from the patient) signal aprobability that a given course or outcome will occur. For example, whenone or more prognostic indicators reach a sufficiently high level insamples obtained from such patients, the level may signal that thepatient is at an increased probability for experiencing morbidity ormortality in comparison to a similar patient exhibiting a lower markerlevel. A level or a change in level of a prognostic indicator, which inturn is associated with an increased probability of morbidity or death,is referred to as being “associated with an increased predisposition toan adverse outcome” in a patient.

The term “correlating” or “relating” as used herein in reference to theuse of markers, refers to comparing the presence or amount of themarker(s) in a patient to its presence or amount in persons known tosuffer from, or known to be at risk of, a given condition; or in personsknown to be free of a given condition. As discussed above, a markerlevel in a patient sample can be compared to a level known to beassociated with a specific diagnosis. The sample's marker level is saidto have been correlated with a diagnosis; that is, the skilled artisancan use the marker level to determine whether the patient suffers from aspecific type diagnosis, and respond accordingly. Alternatively, thesample's marker level can be compared to a marker level known to beassociated with a good outcome (e.g., the absence of disease, etc.). Inpreferred embodiments, a profile of marker levels are correlated to aglobal probability or a particular outcome using ROC curves.

In certain embodiments, the methods described herein comprise thecomparison of an assay result to a corresponding baseline result. Theterm “baseline result” as used herein refers to an assay value that isused as a comparison value (that is, to which a test result iscompared). In practical terms, this means that a marker is measured in asample from a subject, and the result is compared to the baselineresult. A value above the baseline indicates a first likelihood of adiagnosis or prognosis, and a value below the baseline indicates asecond likelihood of a diagnosis or prognosis.

In certain embodiments, a baseline for NGAL (and/or one or more markersrelated thereto) is established, and an NGAL assay result is establishedby performing an assay method that detects NGAL on a sample from apatient. That NGAL result is compared to the NGAL baseline result, and avalue above the baseline indicates worsening renal function, relative toa value below the baseline. Similarly, a value below the baselineindicates improved renal function, relative to a value above thebaseline.

In certain embodiments, a baseline for NGAL (and/or one or more markersrelated thereto) is established, and an NGAL assay result is establishedby performing an assay method that detects NGAL on a sample from apatient. That NGAL result is compared to the NGAL baseline result, and avalue above the baseline indicates an increased mortality risk, relativeto a value below the baseline. Similarly, a value below the baselineindicates a decrease mortality risk, relative to a value above thebaseline.

In other embodiments, a baseline for NGAL (and/or one or more markersrelated thereto) is established, and an NGAL assay result is establishedby performing an assay method that detects NGAL on a sample from apatient. That NGAL result is compared to the NGAL baseline result, and avalue above the baseline indicates worsening renal function, relative toa value below the baseline. Similarly, a value below the baselineindicates improved renal function, relative to a value above thebaseline.

In still other embodiments, a baseline for NGAL (and/or one or moremarkers related thereto) is established, and an NGAL assay result isestablished by performing an assay method that detects NGAL on a samplefrom a patient. That NGAL result is compared to the NGAL baselineresult, and a value above the baseline indicates worsening cardiorenalsyndrome status, relative to a value below the baseline. Similarly, avalue below the baseline indicates improved cardiorenal syndrome status,relative to a value above the baseline.

In yet other embodiments, a natriuretic peptide baseline (for BNP and/orone or more markers related thereto) is established, and a natruireticpeptide assay result is established by performing an assay method thatdetects BNP (and/or one or more markers related thereto) on a samplefrom a patient. That natruiretic peptide assay result is compared to thenatriuretic peptide baseline result, and a value above the baselineindicates worsening cardiorenal syndrome status, relative to a valuebelow the baseline. Similarly, a value below the baseline indicatesimproved cardiorenal syndrome status, relative to a value above thebaseline.

A baseline can be selected in a number of manners well known to those ofskill in the art. For example, data for a marker or markers (e.g.,concentration in a body fluid, such as urine, blood, serum, or plasma)may be obtained from a population of subjects. The population ofsubjects is divided into at least two subpopulations. The firstsubpopulation includes those subjects who have been confirmed as havinga disease, outcome, or, more generally, being in a first conditionstate. For example, this first subpopulation of patients may be thosediagnosed with heart failure, and that suffered from a worsening ofrenal function. For convenience, subjects in this first subpopulationwill be referred to as “diseased,” although in fact, this subpopulationis actually selected for the presence of a particular characteristic ofinterest. The second subpopulation of subjects is formed from thesubjects that do not fall within the first subpopulation. Subjects inthis second set will hereinafter be referred to as “non-diseased.”

A baseline result may then be selected to distinguish between thediseased and non-diseased subpopulation with an acceptable specificityand sensitivity. Changing the baseline merely trades off between thenumber of false positives and the number of false negatives resultingfrom the use of the particular marker under study. The effectiveness ofa test having such an overlap is often expressed using a ROC (ReceiverOperating Characteristic) curve. ROC curves are well known to thoseskilled in the art. The horizontal axis of the ROC curve represents(1-specificity), which increases with the rate of false positives. Thevertical axis of the curve represents sensitivity, which increases withthe rate of true positives. Thus, for a particular cutoff selected, thevalue of (1-specificity) may be determined, and a correspondingsensitivity may be obtained. The area under the ROC curve is a measureof the probability that the measured marker level will allow correctidentification of a disease or condition. Thus, the area under the ROCcurve can be used to determine the effectiveness of the test.

In an alternative, an individual subject may provide their own baseline,in that a temporal change is used to indicate a particular diagnosis orprognosis. For example, one or more markers may be determined at aninitial time to provide one or more baseline results, and then again ata later time, and the change (or lack thereof) in the marker level(s)over time determined. In such embodiments, an increase in the markerfrom the initial time to the second time may be indicative of aparticular prognosis, of a particular diagnosis, etc. Likewise, adecrease in the marker from the initial time to the second time may beindicative of a particular prognosis, of a particular diagnosis, etc. Insuch an embodiment, a plurality of markers need not change in concertwith one another. Temporal changes in one or more markers may also beused together with single time point marker levels compared to apopulation-based baseline.

As discussed herein, the measurement of the level of a single marker maybe augmented by additional markers. For example, other markers relatedto blood pressure regulation, including other natriuretic peptidesand/or their related markers may be used together with, or separatelyfrom, BNP and/or its related markers. Suitable assays include, but arenot limited to, assays that detect ANP, proANP, NT-proANP, CNP,Kininogen, CGRP II, urotensin II, BNP, NT-proBNP, proBNP, calcitoningene related peptide, arg-Vasopressin, Endothelin-1 (and/or Big ET-1),Endothelin-2 (and/or Big ET-2), Endothelin-3 (and/or Big ET-3),procalcitonin, calcyphosine, adrenomedullin, aldosterone, angiotensin 1(and/or angiotensinogen 1), angiotensin 2 (and/or angiotensinogen 2),angiotensin 3 (and/or angiotensinogen 3), Bradykinin, Tachykinin-3,calcitonin, Renin, Urodilatin, and Ghrelin, and/or one or more markersrelated thereto.

Other markers of renal function may also be used in the methodsdescribed herein. These include serum creatinine levels, creatinineclearance rates, cystatin C levels, and glomerular filtration rates.

In addition, subjects suffering from heart failure and renal dysfunctionare often at increased risk for cardiovascular diseases, includingpotentially fatal arrhythmias and acute coronary syndromes. Thus,various subject-derived markers of cardiovascular risk may be includedtogether with NGAL (and/or its related markers) and, where appropriateBNP (and/or its related markers). Suitable subject-derived markersinclude markers related to myocardial injury, markers related tocoagulation and hemostasis, and markers related to inflammation. Inaddition, because of injury to pulmonary tissues occurring in heartfailure, various markers related to pulmonary injury may also beincluded. Exemplary subject-derived markers are provided in thefollowing table:

Marker Classification Neutrophil elastase Pulmonary injury KL-6Pulmonary injury LAMP 3 Pulmonary injury LAMP3 Pulmonary injury LungSurfactant protein A Pulmonary injury Lung Surfactant protein BPulmonary injury Lung Surfactant protein C Pulmonary injury LungSurfactant protein D Pulmonary injury phospholipase D Pulmonary injuryPLA2G5 Pulmonary injury SFTPC Pulmonary injury Myoglobin Tissue injuryE-selectin Tissue injury VEGF Tissue injury EG-VEGF Tissue injuryTroponin I and complexes Myocardial injury Troponin T and complexesMyocardial injury Annexin V Myocardial injury B-enolase Myocardialinjury CK-MB Myocardial injury Glycogen phosphorylase-BB Myocardialinjury Heart type fatty acid binding protein Myocardial injuryPhosphoglyceric acid mutase Myocardial injury S-100ao Myocardial injuryPlasmin Coagulation and hemostasis Thrombin Coagulation and hemostasisAntithrombin-III Coagulation and hemostasis Fibrinogen Coagulation andhemostasis von Willebrand factor Coagulation and hemostasis D-dimerCoagulation and hemostasis PAI-1 Coagulation and hemostasis Protein CCoagulation and hemostasis Soluble Endothelial Protein C Receptor (EPCR)Coagulation and hemostasis TAFI Coagulation and hemostasisFibrinopeptide A Coagulation and hemostasis Plasmin alpha 2 antiplasmincomplex Coagulation and hemostasis Platelet factor 4 Coagulation andhemostasis Platelet-derived growth factor Coagulation and hemostasisP-selectin Coagulation and hemostasis Prothrombin fragment 1 + 2Coagulation and hemostasis B-thromboglobulin Coagulation and hemostasisThrombin antithrombin III complex Coagulation and hemostasisThrombomodulin Coagulation and hemostasis Thrombus Precursor ProteinCoagulation and hemostasis Tissue factor Coagulation and hemostasisTissue factor pathway inhibitor-α Coagulation and hemostasis Tissuefactor pathway inhibitor-β Coagulation and hemostasis APRIL (TNF ligandsuperfamily member 13) Inflammatory CD27 (TNFRSF7) InflammatoryComplement C3a Inflammatory CCL-5 (RANTES) Inflammatory CCL-8 (MCP-2)Inflammatory CCL-16 Inflammatory CCL-19 (macrophage inflammatoryprotein-3β) Inflammatory CCL-20 (MIP-3α) Inflammatory CCL-23 (MIP-3)Inflammatory CXCL-5 (small inducible cytokine B5) Inflammatory CXCL-9(small inducible cytokine B9) Inflammatory CXCL-13 (small induciblecytokine B13) Inflammatory CXCL-16 (small inducible cytokine B16)Inflammatory DPP-II (dipeptidyl peptidase II) Inflammatory DPP-IV(dipeptidyl peptidase W) Inflammatory Glutathione S TransferaseInflammatory HIF 1 ALPHA Inflammatory IL-25 Inflammatory IL-23Inflammatory IL-22 Inflammatory IL-18 Inflammatory IL-13 InflammatoryIL-12 Inflammatory IL-10 Inflammatory IL-1-Beta Inflammatory IL-lraInflammatory IL-4 Inflammatory IL-6 Inflammatory IL-8 InflammatoryLysophosphatidic acid Inflammatory MDA-modified LDL Inflammatory Humanneutrophil elastase Inflammatory C-reactive protein InflammatoryInsulin-like growth factor Inflammatory Inducible nitric oxide synthaseInflammatory Intracellular adhesion molecule Inflammatory Lactatedehydrogenase Inflammatory MCP-1 Inflammatory MMP-1 Inflammatory MMP-2Inflammatory MMP-3 Inflammatory MMP-7 Inflammatory MMP-9 InflammatoryTIMP-1 Inflammatory TIMP-2 Inflammatory TIMP-3 Inflammatory NGALInflammatory n-acetyl aspartate Inflammatory PTEN InflammatoryPhospholipase A2 Inflammatory TNF Receptor Superfamily Member IAInflammatory TNFRSF3 (lymphotoxin β receptor) Inflammatory Transforminggrowth factor beta Inflammatory TREM-1 Inflammatory TREM-1svInflammatory TL-1 (TNF ligand related molecule-1) Inflammatory TL-1aInflammatory Tumor necrosis factor alpha Inflammatory Vascular celladhesion molecule Inflammatory Vascular endothelial growth factorInflammatory cystatin C Inflammatory substance P InflammatoryMyeloperoxidase (MPO) Inflammatory macrophage inhibitory factorInflammatory Fibronectin Inflammatory cardiotrophin 1 InflammatoryHaptoglobin Inflammatory PAPPA Inflammatory s-CD40 ligand InflammatoryHMG-1 (or HMGB1) Inflammatory IL-2 Inflammatory IL-4 Inflammatory IL-11Inflammatory IL-13 Inflammatory IL-18 Inflammatory Eosinophil cationicprotein Inflammatory Mast cell tryptase Inflammatory VCAM InflammatorysICAM-1 Inflammatory TNFα Inflammatory Osteoprotegerin InflammatoryProstaglandin D-synthase Inflammatory Prostaglandin E2 Inflammatory RANKligand Inflammatory RANK (TNFRSF1 1A) Inflammatory HSP-60 InflammatorySerum Amyloid A Inflammatory s-iL 18 receptor Inflammatory S-iL-1receptor Inflammatory s-TNFR1 (P55) Inflammatory s-TNFR2 (P75)Inflammatory sTLR-1 (soluble toll-like receptor-1) Inflammatory sTLR-2Inflammatory sTLR-4 Inflammatory TGF-beta Inflammatory MMP-11Inflammatory Beta NGF Inflammatory CD44 Inflammatory EGF InflammatoryE-selectin Inflammatory Fibronectin Inflammatory RAGE Inflammatory

Various clinical variables may also be utilized as variables in themethods described herein. Examples of such variables include urineoutput levels, age, the presence or absence of one or morecardiovascular risk factors such as diabetes, hypertension, smokingstatus, etc. This list is not meant to be limiting.

Suitable methods for combining markers into a single composite valuethat may be used as if it is a single marker are described in detail inU.S. Provisional Patent Application No. 60/436,392 filed Dec. 24, 2002,PCT application US03/41426 filed Dec. 23, 2003, U.S. patent applicationSer. No. 10/331,127 filed Dec. 27, 2002, and PCT application No.US03/41453, each of which is hereby incorporated by reference in itsentirety, including all tables, figures, and claims. In an alternative,NGAL assay results, natriuretic peptide assay results, and otheroptional test results may be used in an “n-of-m” type of approach. Usinga two marker example of such methods, when either marker above itscorresponding baseline value may signal an increased risk of an adverseoutcome, or a worsening in cardiorenal syndrome status (in n-of-m terms,this is a “1-of-2” result). If both are above the correspondingbaselines (a “2-of-2” result), an even greater worsening in cardiorenalsyndrome status may be indicated.

The sensitivity and specificity of a diagnostic and/or prognostic testdepends on more than just the analytical “quality” of the test—they alsodepend on the definition of what constitutes an abnormal result. Inpractice, Receiver Operating Characteristic curves, or “ROC” curves, aretypically calculated by plotting the value of a variable versus itsrelative frequency in “normal” and “disease” populations. For anyparticular marker, a distribution of marker levels for subjects with andwithout a “disease” will likely overlap. Under such conditions, a testdoes not absolutely distinguish normal from disease with 100% accuracy,and the area of overlap indicates where the test cannot distinguishnormal from disease. A threshold is selected, above which (or belowwhich, depending on how a marker changes with the disease) the test isconsidered to be abnormal and below which the test is considered to benormal. The area under the ROC curve is a measure of the probabilitythat the perceived measurement will allow correct identification of acondition. ROC curves can be used even when test results don'tnecessarily give an accurate number. As long as one can rank results,one can create an ROC curve. For example, results of a test on “disease”samples might be ranked according to degree (say 1=low, 2=normal, and3=high). This ranking can be correlated to results in the “normal”population, and a ROC curve created. These methods are well known in theart. See, e.g., Hanley et al., Radiology 143: 29-36 (1982).

Measures of test accuracy may also be obtained as described in Fischeret al., Intensive Care Med. 29: 1043-51, 2003, and used to determine theeffectiveness of a given marker or panel of markers. These measuresinclude sensitivity and specificity, predictive values, likelihoodratios, diagnostic odds ratios, and ROC curve areas. As discussed above,preferred tests and assays exhibit one or more of the following resultson these various measures.

Preferably, a baseline is chosen to exhibit at least about 70%sensitivity, more preferably at least about 80% sensitivity, even morepreferably at least about 85% sensitivity, still more preferably atleast about 90% sensitivity, and most preferably at least about 95%sensitivity, combined with at least about 70% specificity, morepreferably at least about 80% specificity, even more preferably at leastabout 85% specificity, still more preferably at least about 90%specificity, and most preferably at least about 95% specificity. Inparticularly preferred embodiments, both the sensitivity and specificityare at least about 75%, more preferably at least about 80%, even morepreferably at least about 85%, still more preferably at least about 90%,and most preferably at least about 95%. The term “about” in this contextrefers to +/−5% of a given measurement.

In other embodiments, a positive likelihood ratio, negative likelihoodratio, odds ratio, or hazard ratio is used as a measure of a test'sability to predict risk or diagnose a disease. In the case of a positivelikelihood ratio, a value of 1 indicates that a positive result isequally likely among subjects in both the “diseased” and “control”groups; a value greater than 1 indicates that a positive result is morelikely in the diseased group; and a value less than 1 indicates that apositive result is more likely in the control group. In the case of anegative likelihood ratio, a value of 1 indicates that a negative resultis equally likely among subjects in both the “diseased” and “control”groups; a value greater than 1 indicates that a negative result is morelikely in the test group; and a value less than 1 indicates that anegative result is more likely in the control group. In certainpreferred embodiments, markers and/or marker panels are preferablyselected to exhibit a positive or negative likelihood ratio of at leastabout 1.5 or more or about 0.67 or less, more preferably at least about2 or more or about 0.5 or less, still more preferably at least about 5or more or about 0.2 or less, even more preferably at least about 10 ormore or about 0.1 or less, and most preferably at least about 20 or moreor about 0.05 or less. The term “about” in this context refers to +/−5%of a given measurement.

In the case of an odds ratio, a value of 1 indicates that a positiveresult is equally likely among subjects in both the “diseased” and“control” groups; a value greater than 1 indicates that a positiveresult is more likely in the diseased group; and a value less than 1indicates that a positive result is more likely in the control group. Anodds ratio of at least about 1.25 or about 0.8 or less can provideacceptable performance. In certain preferred embodiments, markers and/ormarker panels are preferably selected to exhibit an odds ratio of atleast about 2 or more or about 0.5 or less, more preferably at leastabout 3 or more or about 0.33 or less, still more preferably at leastabout 4 or more or about 0.25 or less, even more preferably at leastabout 5 or more or about 0.2 or less, and most preferably at least about10 or more or about 0.1 or less. The term “about” in this context refersto +/−5% of a given measurement.

In the case of a hazard ratio, a value of 1 indicates that the relativerisk of an endpoint (e.g., death) is equal in both the “diseased” and“control” groups; a value greater than 1 indicates that the risk isgreater in the diseased group; and a value less than 1 indicates thatthe risk is greater in the control group. In certain preferredembodiments, markers and/or marker panels are preferably selected toexhibit a hazard ratio of at least about 1.1 or more or about 0.91 orless, more preferably at least about 1.25 or more or about 0.8 or less,still more preferably at least about 1.5 or more or about 0.67 or less,even more preferably at least about 2 or more or about 0.5 or less, andmost preferably at least about 2.5 or more or about 0.4 or less. Theterm “about” in this context refers to +/−5% of a given measurement.

Numerous methods and devices are well known to the skilled artisan forthe detection and analysis of the markers of the instant invention. Withregard to polypeptides or proteins in patient test samples, immunoassaydevices and methods are often used. See, e.g., U.S. Pat. Nos. 6,143,576;6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615;5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792,each of which is hereby incorporated by reference in its entirety,including all tables, figures and claims. These devices and methods canutilize labeled molecules in various sandwich, competitive, ornon-competitive assay formats, to generate a signal that is related tothe presence or amount of an analyte of interest. Additionally, certainmethods and devices, such as biosensors and optical immunoassays, may beemployed to determine the presence or amount of analytes without theneed for a labeled molecule. See, e.g., U.S. Pat. Nos. 5,631,171; and5,955,377, each of which is hereby incorporated by reference in itsentirety, including all tables, figures and claims. One skilled in theart also recognizes that robotic instrumentation including but notlimited to Beckman Access, Abbott AxSym, Roche ElecSys, Dade BehringStratus systems are among the immunoassay analyzers that are capable ofperforming the immunoassays taught herein.

Preferably the markers are analyzed using an immunoassay, and mostpreferably sandwich immunoassay, although other methods are well knownto those skilled in the art (for example, the measurement of marker RNAlevels). The presence or amount of a marker is generally determinedusing antibodies specific for each marker and detecting specificbinding. Any suitable immunoassay may be utilized, for example,enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs),competitive binding assays, and the like. Specific immunological bindingof the antibody to the marker can be detected directly or indirectly.Biological assays such as immunoassays require methods for detection,and one of the most common methods for quantitation of results is toconjugate an enzyme, fluorophore or other molecule to form anantibody-label conjugate. Detectable labels may include molecules thatare themselves detectable (e.g., fluorescent moieties, electrochemicallabels, metal chelates, etc.) as well as molecules that may beindirectly detected by production of a detectable reaction product(e.g., enzymes such as horseradish peroxidase, alkaline phosphatase,etc.) or by a specific binding molecule which itself may be detectable(e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene,phenylarsenate, ssDNA, dsDNA, etc.). Particularly preferred detectablelabels are fluorescent latex particles such as those described in U.S.Pat. Nos. 5,763,189, 6,238,931, and 6,251,687; and InternationalPublication WO95/08772, each of which is hereby incorporated byreference in its entirety. Exemplary conjugation to such particles isdescribed hereinafter. Direct labels include fluorescent or luminescenttags, metals, dyes, radionuclides, and the like, attached to theantibody. Indirect labels include various enzymes well known in the art,such as alkaline phosphatase, horseradish peroxidase and the like.

The use of immobilized antibodies specific for the markers is alsocontemplated by the present invention. The term “solid phase” as usedherein refers to a wide variety of materials including solids,semi-solids, gels, films, membranes, meshes, felts, composites,particles, papers and the like typically used by those of skill in theart to sequester molecules. The solid phase can be non-porous or porous.Suitable solid phases include those developed and/or used as solidphases in solid phase binding assays. See, e.g., chapter 9 ofImmunoassay, E. P. Dianiandis and T. K. Christopoulos eds., AcademicPress: New York, 1996, hereby incorporated by reference. Examples ofsuitable solid phases include membrane filters, cellulose-based papers,beads (including polymeric, latex and paramagnetic particles), glass,silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGAgels, SPOCC gels, and multiple-well plates. See, e.g., Leon et al.,Bioorg. Med. Chem. Lett. 8: 2997, 1998; Kessler et al., Agnew. Chem.Int. Ed. 40: 165, 2001; Smith et al., J. Comb. Med. 1: 326, 1999; Orainet al., Tetrahedron Lett. 42: 515, 2001; Papanikos et al., J. Am. Chem.Soc. 123: 2176, 2001; Gottschling et al., Bioorg. Med. Chem. Lett. 11:2997, 2001. The antibodies could be immobilized onto a variety of solidsupports, such as magnetic or chromatographic matrix particles, thesurface of an assay place (such as microtiter wells), pieces of a solidsubstrate material or membrane (such as plastic, nylon, paper), and thelike. An assay strip could be prepared by coating the antibody or aplurality of antibodies in an array on solid support. This strip couldthen be dipped into the test sample and then processed quickly throughwashes and detection steps to generate a measurable signal, such as acolored spot. When multiple assays are being performed, a plurality ofseparately addressable locations, each corresponding to a differentmarker and comprising antibodies that bind the appropriate marker, canbe provided on a single solid support. The term “discrete” as usedherein refers to areas of a surface that are non-contiguous. That is,two areas are discrete from one another if a border that is not part ofeither area completely surrounds each of the two areas. The term“independently addressable” as used herein refers to discrete areas of asurface from which a specific signal may be obtained.

For separate or sequential assay of markers, suitable apparatusesinclude clinical laboratory analyzers such as the ElecSys (Roche), theAxSym (Abbott), the Access (Beckman), the ADVIA® CENTAUR® (Bayer)immunoassay systems, the NICHOLS ADVANTAGE® (Nichols Institute)immunoassay system, etc. Preferred apparatuses perform simultaneousassays of a plurality of markers using a single test device.Particularly useful physical formats comprise surfaces having aplurality of discrete, addressable locations for the detection of aplurality of different analytes. Such formats include proteinmicroarrays, or “protein chips” (see, e.g., Ng and flag, J. Cell Mol.Med. 6: 329-340 (2002)) and certain capillary devices (see, e.g., U.S.Pat. No. 6,019,944). In these embodiments, each discrete surfacelocation may comprise antibodies to immobilize one or more analyte(s)(e.g., a marker) for detection at each location. Surfaces mayalternatively comprise one or more discrete particles (e.g.,microparticles or nanoparticles) immobilized at discrete locations of asurface, where the microparticles comprise antibodies to immobilize oneanalyte (e.g., a marker) for detection.

Preferred assay devices of the present invention will comprise, for oneor more assays, a first antibody conjugated to a solid phase and asecond antibody conjugated to a signal development element. Such assaydevices are configured to perform a sandwich immunoassay for one or moreanalytes. These assay devices will preferably further comprise a sampleapplication zone, and a flow path from the sample application zone to asecond device region comprising the first antibody conjugated to a solidphase.

Flow of a sample in an assay device along the flow path may be drivenpassively (e.g., by capillary, hydrostatic, or other forces that do notrequire further manipulation of the device once sample is applied),actively (e.g., by application of force generated via mechanical pumps,electroosmotic pumps, centrifugal force, increased air pressure, etc.),or by a combination of active and passive driving forces. Mostpreferably, sample applied to the sample application zone will contactboth a first antibody conjugated to a solid phase and a second antibodyconjugated to a signal development element along the flow path (sandwichassay format). Additional elements, such as filters to separate plasmaor serum from blood, mixing chambers, etc., may be included as requiredby the artisan. Exemplary devices are described in Chapter 41, entitled“Near Patient Tests: Triage® Cardiac System,” in The ImmunoassayHandbook, 2^(nd) ed., David Wild, ed., Nature Publishing Group, 2001,which is hereby incorporated by reference in its entirety.

The analysis of markers could be carried out in a variety of physicalformats as well. For example, the use of microtiter plates or automationcould be used to facilitate the processing of large numbers of testsamples. Alternatively, single sample formats could be developed tofacilitate immediate treatment and diagnosis in a timely fashion, forexample, in ambulatory transport or emergency room settings.

In another embodiment, the present invention provides a kit for theanalysis of markers. Such a kit preferably comprises devises andreagents for the analysis of at least one test sample and instructionsfor performing the assay(s) of interest. Optionally the kits may containone or more means for using information obtained from immunoassaysperformed for a marker panel to rule in or out certain diagnoses orprognoses. Other measurement strategies applicable to the methodsdescribed herein include chromatography (e.g., HPLC), mass spectrometry,receptor-based assays, and combinations of the foregoing.

The term “antibody” as used herein refers to a peptide or polypeptidederived from, modeled after or substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof,capable of specifically binding an antigen or epitope. See, e.g.Fundamental Immunology, 3^(rd) Edition, W. E. Paul, ed., Raven Press,N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush(1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

Preferably, an antibody is selected that specifically binds a marker ofinterest. The term “specifically binds” is not intended to indicate thatan antibody binds exclusively to its intended target. Rather, anantibody “specifically binds” if its affinity for its intended target isabout 5-fold greater when compared to its affinity for a non-targetmolecule. Preferably the affinity of the antibody will be at least about5 fold, preferably 10 fold, more preferably 25-fold, even morepreferably 50-fold, and most preferably 100-fold or more, greater for atarget molecule than its affinity for a non-target molecule. Inpreferred embodiments, Specific binding between an antibody or otherbinding agent and an antigen means a binding affinity of at least 10⁶M⁻¹. Preferred antibodies bind with affinities of at least about 10⁷M⁻¹, and preferably between about 10⁸ M⁻¹ to about 10⁹ M⁻¹, about 10⁹M⁻¹ to about 10¹⁰ M⁻¹, or about 10¹⁰ M⁻¹ to about 10¹¹ M⁻¹.

Affinity is calculated as K_(d)=k_(off)/k_(on) (k_(off) is thedissociation rate constant, k_(on) is the association rate constant andK_(d) is the equilibrium constant. Affinity can be determined atequilibrium by measuring the fraction bound (r) of labeled ligand atvarious concentrations (c). The data are graphed using the Scatchardequation: r/c=K(n-r):

-   -   where

r=moles of bound ligand/mole of receptor at equilibrium;

c=free ligand concentration at equilibrium;

K=equilibrium association constant; and

n=number of ligand binding sites per receptor molecule

By graphical analysis, r/c is plotted on the Y-axis versus r on theX-axis thus producing a Scatchard plot. The affinity is the negativeslope of the line. k_(off) can be determined by competing bound labeledligand with unlabeled excess ligand (see, e.g., U.S. Pat. No.6,316,409). The affinity of a targeting agent for its target molecule ispreferably at least about 1×10⁻⁶ moles/liter, is more preferably atleast about 1×10⁻⁷ moles/liter, is even more preferably at least about1×10⁻⁸ moles/liter, is yet even more preferably at least about 1×10⁻⁹moles/liter, and is most preferably at least about 1×10⁻¹⁰ moles/liter.Antibody affinity measurement by Scatchard analysis is well known in theart. See, e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelsonand Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.

The generation and selection of antibodies may be accomplished severalways. For example, one way is to purify polypeptides of interest or tosynthesize the polypeptides of interest using, e.g., solid phase peptidesynthesis methods well known in the art. See, e.g., Guide to ProteinPurification, Murray P. Deutcher, ed., Meth. Enzymol. Vol 182 (1990);Solid Phase Peptide Synthesis, Greg B. Fields ed., Meth. Enzymol. Vol289 (1997); Kiso et al., Chem. Pharm. Bull. (Tokyo) 38: 1192-99, 1990;Mostafavi et al., Biomed. Pept. Proteins Nucleic Acids 1: 255-60, 1995;Fujiwara et al., Chem. Pharm. Bull. (Tokyo) 44: 1326-31, 1996. Theselected polypeptides may then be injected, for example, into mice orrabbits, to generate polyclonal or monoclonal antibodies. One skilled inthe art will recognize that many procedures are available for theproduction of antibodies, for example, as described in Antibodies, ALaboratory Manual, Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988), Cold Spring Harbor, N.Y. One skilled in the art willalso appreciate that binding fragments or Fab fragments which mimicantibodies can also be prepared from genetic information by variousprocedures (Antibody Engineering: A Practical Approach, Borrebaeck, C.,ed., 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920(1992)).

In addition, numerous publications have reported the use of phagedisplay technology to produce and screen libraries of polypeptides forbinding to a selected target. See, e.g., Cwirla et al., Proc. Natl.Acad. Sci. USA 87, 6378-82, 1990; Devlin et al., Science 249, 404-6,1990, Scott and Smith, Science 249, 386-88, 1990; and Ladner et al.,U.S. Pat. No. 5,571,698. A basic concept of phage display methods is theestablishment of a physical association between DNA encoding apolypeptide to be screened and the polypeptide. This physicalassociation is provided by the phage particle, which displays apolypeptide as part of a capsid enclosing the phage genome which encodesthe polypeptide. The establishment of a physical association betweenpolypeptides and their genetic material allows simultaneous massscreening of very large numbers of phage bearing different polypeptides.Phage displaying a polypeptide with affinity to a target bind to thetarget and these phage are enriched by affinity screening to the target.The identity of polypeptides displayed from these phage can bedetermined from their respective genomes. Using these methods apolypeptide identified as having a binding affinity for a desired targetcan then be synthesized in bulk by conventional means. See, e.g., U.S.Pat. No. 6,057,098, which is hereby incorporated in its entirety,including all tables, figures, and claims.

The antibodies that are generated by these methods may then be selectedby first screening for affinity and specificity with the purifiedpolypeptide of interest and, if required, comparing the results to theaffinity and specificity of the antibodies with polypeptides that aredesired to be excluded from binding. The screening procedure can involveimmobilization of the purified polypeptides in separate wells ofmicrotiter plates. The solution containing a potential antibody orgroups of antibodies is then placed into the respective microtiter wellsand incubated for about 30 min to 2 h. The microtiter wells are thenwashed and a labeled secondary antibody (for example, an anti-mouseantibody conjugated to alkaline phosphatase if the raised antibodies aremouse antibodies) is added to the wells and incubated for about 30 minand then washed. Substrate is added to the wells and a color reactionwill appear where antibody to the immobilized polypeptide(s) arepresent.

The antibodies so identified may then be further analyzed for affinityand specificity in the assay design selected. In the development ofimmunoassays for a target protein, the purified target protein acts as astandard with which to judge the sensitivity and specificity of theimmunoassay using the antibodies that have been selected. Because thebinding affinity of various antibodies may differ; certain antibodypairs (e.g., in sandwich assays) may interfere with one anothersterically, etc., assay performance of an antibody may be a moreimportant measure than absolute affinity and specificity of an antibody.

Those skilled in the art will recognize that many approaches can betaken in producing antibodies or binding fragments and screening andselecting for affinity and specificity for the various polypeptides, butthese approaches do not change the scope of the invention.

EXAMPLES

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

Example 1 Biochemical Analyses

Markers were measured using standard immunoassay techniques. Thesetechniques involve the use of antibodies to specifically bind theanalyte(s) of interest Immunoassays were performed using TECAN GenesisRSP 200/8 or Perkin Elmer Minitrak Workstations, or using microfluidicdevices manufactured at Biosite Incorporated essentially as described inWO98/43739, WO98/08606, WO98/21563, and WO93/24231. Analytes may bemeasured using a sandwich immunoassay or using a competitive immunoassayas appropriate, depending on the characteristics and concentration rangeof the analyte of interest.

The assays were calibrated using purified proteins (that is either thesame as or related to the selected analyte, and that can be detected inthe assay) diluted gravimetrically into EDTA plasma treated in the samemanner as the sample population specimens. Endogenous levels of theanalyte present in the plasma prior to addition of the purified markerprotein was measured and taken into account in assigning the markervalues in the calibrators. When necessary to reduce endogenous levels inthe calibrators, the endogenous analyte was stripped from the plasmausing standard immunoaffinity methods. Calibrators were assayed in thesame manner as the sample population specimens, and the resulting dataused to construct a “dose-response” curve (assay signal as a function ofanalyte concentration), which may be used to determine analyteconcentrations from assay signals obtained from subject specimens.

Individual assays were configured to bind the following markers, andresults are reported in the following examples using the followingunits: BNP—pg/mL; NGAL—ng/mL; cystatin C—μg/mL.

Example 2 Microtiter Plate-Based Biochemical Analyses

For the sandwich immunoassay in microtiter plates, a monoclonal antibodydirected against a selected analyte was biotinylated usingN-hydroxysuccinimide biotin (NHS-biotin) at a ratio of about 5NHS-biotin moieties per antibody. The antibody-biotin conjugate was thenadded to wells of a standard avidin 384 well microtiter plate, andantibody conjugate not bound to the plate was removed. This formed the“anti-marker” in the microtiter plate. Another monoclonal antibodydirected against the same analyte was conjugated to alkalinephosphatase, for example using succinimidyl4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC) andN-succinimidyl 3-[2-pyridyldithio]propionate (SPDP) (Pierce, Rockford,Ill.).

Biotinylated antibodies were pipetted into microtiter plate wellspreviously coated with avidin and incubated for 60 mM. The solutioncontaining unbound antibody was removed, and the wells washed with awash buffer, consisting of 20 mM borate (pH 7.42) containing 150 mMNaCl, 0.1% sodium azide, and 0.02% Tween-20. The plasma samples (10 μL)containing added HAMA inhibitors were pipeted into the microtiter platewells, and incubated for 60 min. The sample was then removed and thewells washed with a wash buffer. The antibody-alkaline phosphataseconjugate was then added to the wells and incubated for an additional 60min, after which time, the antibody conjugate was removed and the wellswashed with a wash buffer. A substrate, (AttoPhos®, Promega, andMadison, Wis.) was added to the wells, and the rate of formation of thefluorescent product is related to the concentration of the analyte inthe sample tested.

Example 3 Microfluidic Device-Based Biochemical Analyses

Immunoassays were performed using microfluidic devices essentially asdescribed in Chapter 41, entitled “Near Patient Tests: Triage® CardiacSystem,” in The Immunoassay Handbook, 2^(nd) ed., David Wild, ed.,Nature Publishing Group, 2001.

For sandwich immunoassays, a plasma sample was added to the microfluidicdevice that contains all the necessary assay reagents, including HAMAinhibitors, in dried form. The plasma passed through a filter to removeparticulate matter. Plasma enterd a “reaction chamber” by capillaryaction. This reaction chamber contained fluorescent latexparticle-antibody conjugates (hereafter called FETL-antibody conjugates)appropriate to an analyte of interest, and may contain FETL-antibodyconjugates to several selected analytes. The FETL-antibody conjugatesdissolved into the plasma to form a reaction mixture, which was held inthe reaction chamber for an incubation period (about a minute) to allowthe analyte(s) of interest in the plasma to bind to the antibodies.After the incubation period, the reaction mixture moved down thedetection lane by capillary action. Antibodies to the analyte(s) ofinterest were immobilized in discrete capture zones on the surface of a“detection lane.” Analyte/antibody-FETL complexes formed in the reactionchamber were captured on an appropriate detection zone to form asandwich complex, while unbound FETL-antibody conjugates were washedfrom the detection lane into a waste chamber by excess plasma. Theamount of analyte/antibody-FETL complex bound on a capture zone wasquantified with a fluorometer (Triage® MeterPlus, Biosite Incorporated)and was related to the amount of the selected analyte in the plasmaspecimen.

Example 4 Assigning a Risk of an Adverse Outcome to a Subject Sufferingfrom Heart Failure

Samples were obtained from individuals presenting to the emergencydepartment of a hospital for symptoms of congestive heart failure andthat were subsequently treated for congestive heart failure, either inthe emergency department or as an inpatient following admission to thehospital. Exclusion criteria were: less than 18 years of age, current MIor ACS with ST elevation of 1 mm or greater, renal failure requiringdialysis, hemodialysis within the last month, or a baseline BNP of 100pg/mL or less. 90 day follow-up was by telephone, chart review, or mail.

From this population, individuals were selected such that 50 individualsfell into the following two possible groups: a serum creatinine (“sCr”)at presentation of <1.6 mg/dL, and a serum creatinine≧1.6 mg/dL.Possible outcomes examined were mortality within 90 days; an unplannedvisit to the hospital for CHF or other cardiac event within 90 days(referred to collectively below as a “Cardiac Event”); an unplannedvisit for a non-cardiac event within 90 days; or no such event within 90days.

The following markers were measured in EDTA plasma samples: cystatin C,NGAL, and BNP. Reported values are cystatin C: μg/mL; NGAL: ng/mL; andBNP: pg/mL. The following are the descriptive statistics obtained. Inthese tables, the “event” group represents individuals experiencingmortality or an unplanned visit to the hospital for a Cardiac Event, ineach case within 90 days.

sCr < 1.6, Event sCr ≧ 1.6, Event CYSC NGAL BNP CYSC NGAL BNP n 15 15 15n 15 15 15 Median 1.29 94 370 Median 2.17 148 879 Mean 1.45 103 546 Mean2.42 212 1328 Min 0.90 0 78 Min 1.088 0 113 Max 2.39 313 2226 Max 3.481233 4369 Stdev 0.40 75 549 Stdev 0.80 292 1252

sCr < 1.6, No Event sCr ≧ 1.6, No Event CYSC NGAL BNP CYSC NGAL BNP n 1414 14 n 14 14 14 Median 1.25 69 281 Median 2.30 121 680 Mean 1.44 93 356Mean 2.18 136 612 Min 0.76 0 78 Min 0.95 0 73 Max 3.09 473 1386 Max 3.32333 1504 Stdev 0.70 123 316 Stdev 0.84 93 433

Any sCr, Event Any sCr, No Event CYSC NGAL BNP CYSC NGAL BNP n 30 30 30n 28 28 28 Median 1.72 127 520 Median 1.46 99 328 Mean 1.93 158 937 Mean1.81 114 484 Min 0.90 0 78 Min 0.76 0 73 Max 3.48 1233 4369 Max 3.32 4731504 Stdev 0.79 217 1030 Stdev 0.85 109 394

sCr < 1.6, 90 d mortality sCr ≧ 1.6, 90 d mortality CYSC NGAL BNP CYSCNGAL BNP n 3 3 3 n 4 4 4 Median 1.70 176 519 Median 2.38 111 902 Mean1.79 208 1027 Mean 2.37 114 1119 Min 1.29 136 337 Min 1.74 0 238 Max2.39 313 2226 Max 2.98 232 2434 Stdev 0.56 93 1042 Stdev 0.59 131 963

Any sCr, 90 d mortality CYSC NGAL BNP n 7 7 7 Median 2.01 176 599 Mean2.12 154 1080 Min 1.29 0 238 Max 2.98 313 2434 Stdev 0.61 119 910

ROC analysis was performed to determine the ability of these markers topredict outcome, as shown in the following tables. In each case, the“diseased” group refers to those suffering from mortality or a CardiacEvent (as indicated in the table), and “nondiseased” refers to the noevent group. “Sense” indicates whether the marker is increasing ordecreasing with “disease.” P values were calculated using a one-sample Ztest.

Cystatin C Mortality Cardiac Event sCR <1.6 ≧1.6 Any <1.6 ≧1.6 any ROCArea 0.762 0.571 0.658 0.548 0.623 0.543 N(diseased) 14 14 28 14 14 28N(nondiseased) 3 4 7 12 11 23 p value 0.018 0.306 0.040 0.340 0.1450.298 Sense Increasing Increasing Increasing Increasing IncreasingIncreasing

NGAL Mortality Cardiac Event sCR <1.6 ≧1.6 Any <1.6 ≧1.6 any ROC Area0.881 0.571 0.628 0.530 0.643 0.557 N(diseased) 14 14 28 14 14 28N(nondiseased) 3 4 7 12 11 23 p value <0.001 0.356 0.172 0.401 0.1030.242 Sense Increasing Increasing Increasing Increasing IncreasingIncreasing

BNP Mortality Cardiac Event sCR <1.6 ≧1.6 Any <1.6 ≧1.6 any ROC Area0.857 0.661 0.730 0.554 0.656 0.590 N(diseased) 14 14 28 14 14 28N(nondiseased) 3 4 7 12 11 23 p value <0.001 0.174 0.014 0.330 0.0900.135 Sense Increasing Increasing Increasing Increasing IncreasingIncreasing

These results indicate that cystatin C, NGAL, and BNP are eachsignificant predictors of mortality, particularly in combination with alow initial serum creatinine level.

Using this data, odds ratios were calculated for the low serum creatinegroup, using the median measured level of each marker as a threshold:

Odds ratio for mortality Marker or cardiac event Cystatin C 1.5 NGAL 1.5BNP 2.7 Any one marker elevated 4.9

Dividing the high serum creatinine group into tertiles, odds ratios werecalculated comparing the first tertile to the third tertile:

Odds ratio for mortality Marker or cardiac event Cystatin C 2.3 NGAL 3.5BNP 2.3 Any one marker in 3^(rd) tertile 3.7

These results demonstrate that each marker can be used to assign arelative risk of an adverse outcome to individuals suffering from heartfailure, and that multiple marker strategies that combine two or more ofNGAL, cystatin C, BNP, and serum creatinine can improve this ability toassign risk.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The examples providedherein are representative of preferred embodiments, are exemplary, andare not intended as limitations on the scope of the invention.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

1-22. (canceled)
 23. A method of monitoring cardiorenal syndrome apatient, comprising: (a) performing an assay method that detects NGAL ona body fluid sample obtained from said patient, thereby providing anNGAL assay result; (b) performing an assay method that detects one ormore of BNP, NT-proBNP and proBNP on the same or a different body fluidsample obtained from said patient, thereby providing a natriureticpeptide assay result; and (c) relating the NGAL assay result and thenatriuretic peptide assay result to the patient's cardiorenal syndromestatus by (i) comparing said NGAL assay result to a baseline NGALresult, and assigning a worsening cardiorenal syndrome status to saidpatient when said NGAL assay result is greater than said baseline NGALresult, relative to a status assigned when said NGAL assay result isless than said baseline NGAL result, or by assigning an improvingcardiorenal syndrome status to said patient when said NGAL assay resultis less than said baseline NGAL result, relative to a status assignedwhen said NGAL assay result is greater than said baseline NGAL result,and (ii) comparing said natriuretic peptide assay result to a baselinenatriuretic peptide result, and assigning a worsening cardiorenalsyndrome status to said patient when said natriuretic peptide assayresult is greater than said baseline natriuretic peptide result,relative to a status assigned when said natriuretic peptide assay resultis less than said baseline natriuretic peptide result, or by assigningan improving cardiorenal syndrome status to said patient when saidnatriuretic peptide assay result is less than said baseline natriureticpeptide result, relative to a status assigned when said natriureticpeptide assay result is greater than said baseline natriuretic peptideresult.
 24. A method according to claim 23, wherein said baseline NGALresult is determined by performing an assay method that detects NGAL ona body fluid sample obtained from said patient at a time earlier thanthe time at which the body fluid sample used to provide said NGAL assayresult was obtained.
 25. A method according to claim 23, wherein saidbaseline natriuretic peptide result is determined by performing an assaymethod that detects one or more of BNP, NT-proBNP and proBNP on a bodyfluid sample obtained from said patient at a time earlier than the timeat which the body fluid sample used to provide said natriuretic peptideassay result was obtained.
 26. A method according to claim 23, whereinsaid baseline NGAL result is determined from a population of subjectssuffering from cardiorenal syndrome, and said baseline NGAL result isselected to separate said population into a first subpopulation having aworsening cardiorenal syndrome status relative to a secondsubpopulation.
 27. A method according to claim 23, wherein said baselinenatriuretic peptide result is determined from a population of subjectssuffering from cardiorenal syndrome, and said baseline natriureticpeptide result is selected to separate said population into a firstsubpopulation having an worsening cardiorenal syndrome relative to asecond subpopulation.
 28. A method according to claim 26, wherein saidbaseline NGAL result separates said first subpopulation from said secondsubpopulation with an odds ratio of at least 2 or more or 0.5 or less.29. A method according to claim 26, wherein said baseline NGAL resultseparates said first subpopulation from said second subpopulation withan odds ratio of at least 3 or more or 0.33 or less.
 30. A methodaccording to claim 27, wherein said baseline natriuretic peptide resultseparates said first subpopulation from said second subpopulation withan odds ratio of at least 2 or more or 0.5 or less.
 31. A methodaccording to claim 27, wherein said baseline natriuretic peptide resultseparates said first subpopulation from said second subpopulation withan odds ratio of at least 3 or more or 0.33 or less.
 32. A methodaccording to claim 23, wherein the assay method that detects NGALcomprises performing an immunoassay that detects NGAL, and the assaymethod that detects one or more of BNP, NT-proBNP and proBNP comprisesperforming an immunoassay that detects one or more of BNP, NT-proBNP andproBNP.
 33. A method according to claim 23, wherein the body fluidsample is selected from the group consisting of urine, blood, serum, andplasma. 34-48. (canceled)